The ability to target HER2-driven breast cancers using anticancer agents with mechanisms of actions that are independent of the HER2 cellular growth function, represents a powerful new tool to circumvent drug resistance experienced with traditional HER2-targeted therapies. We have discovered that manipulation of the balance between the DNA repair response and apoptosis pathways provides an alternative strategy to induce targeted apoptosis in HER2-amplified breast cancers. Preliminary work has determined that triplex DNA formed at chromosomal sites by endogenous triplex-forming oligonucleotides (TFOs) can induce apoptosis in human cells. Importantly, apoptosis induction occurs only in response to formation of multiple triplex structures; and not one or two, since the nucleotide excision repair pathway is capable of efficiently repairing a low level of damage. Triplex DNA can be created when TFOs bind as third strands within the major groove of duplex DNA at specific polypurine stretches. HER2-positive breast cancers can have upwards of 25-50 copies of the HER2 gene. Moreover this gene contains several polypurine sites that are conducive to triplex formation. This supplies multiple potential triplex target sites within the malignant cells and creates a distinction between tumor cells and normal cells, which lack HER2 gene amplification. As a result, the level of toxicity induced by HER2-targeted TFOs in normal tissues should be low. This creates the foundation for a new therapeutic alternative in the treatment of HER2-positive breast cancers. We have already designed sequence-specific TFOs, which will bind to their target HER2 sequence, create multiple triplex DNA structures, and specifically activate apoptosis in tumor cells. We will evaluate the efficacy of these molecules using cell growth, clonogenic and apoptosis assays. We will examine the mechanisms involved in triplex-induced apoptosis, with emphasis on components of the DNA damage response pathway that provide crosstalk with apoptosis pathways, and establish whether the mechanisms involved are independent of HER2 cellular function. We will also investigate the potential for gene-targeted apoptosis to inhibit the growth and metastasis of HER2-positive breast cancers in an athymic nude mouse model. Targeting of the HER2 gene on a genomic level using DNA-binding molecules to induce triplex structures provides a novel therapeutic option for the treatment of HER2-positive breast cancers. This approach offers an alternative method to target HER2 by manipulating the cell's DNA damage response machinery, and thus represents a new paradigm for gene-targeted drugs.